CN109668881A - Alkaline phosphatase portable detection reagent box and its application based on temperature change - Google Patents
Alkaline phosphatase portable detection reagent box and its application based on temperature change Download PDFInfo
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- CN109668881A CN109668881A CN201910099989.9A CN201910099989A CN109668881A CN 109668881 A CN109668881 A CN 109668881A CN 201910099989 A CN201910099989 A CN 201910099989A CN 109668881 A CN109668881 A CN 109668881A
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- Prior art keywords
- alkaline phosphatase
- detection reagent
- concentration
- reagent box
- portable
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- 102000002260 Alkaline Phosphatase Human genes 0.000 title claims abstract description 82
- 108020004774 Alkaline Phosphatase Proteins 0.000 title claims abstract description 82
- 238000001514 detection method Methods 0.000 title claims abstract description 75
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 46
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 claims abstract description 48
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 40
- 239000002055 nanoplate Substances 0.000 claims abstract description 21
- 229960003638 dopamine Drugs 0.000 claims abstract description 20
- 239000011734 sodium Substances 0.000 claims abstract description 17
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 17
- 239000007853 buffer solution Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 25
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 24
- 239000000872 buffer Substances 0.000 claims description 24
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 19
- 238000012360 testing method Methods 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 12
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 claims description 2
- 102000013563 Acid Phosphatase Human genes 0.000 claims description 2
- 108010051457 Acid Phosphatase Proteins 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 241000209094 Oryza Species 0.000 claims 2
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 239000007795 chemical reaction product Substances 0.000 abstract description 2
- 238000011896 sensitive detection Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 6
- 229910001425 magnesium ion Inorganic materials 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 6
- 238000006555 catalytic reaction Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 229920001690 polydopamine Polymers 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- 108010074051 C-Reactive Protein Proteins 0.000 description 3
- 102100032752 C-reactive protein Human genes 0.000 description 3
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 3
- 102100022624 Glucoamylase Human genes 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- -1 DOPA Amine Chemical class 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 230000030609 dephosphorylation Effects 0.000 description 2
- 238000006209 dephosphorylation reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 description 2
- 229960004502 levodopa Drugs 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- GDSOZVZXVXTJMI-SNAWJCMRSA-N (e)-1-methylbut-1-ene-1,2,4-tricarboxylic acid Chemical compound OC(=O)C(/C)=C(C(O)=O)\CCC(O)=O GDSOZVZXVXTJMI-SNAWJCMRSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000012123 point-of-care testing Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Pathology (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Plasma & Fusion (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The alkaline phosphatase portable detection reagent box and its application, alkaline phosphatase portable detection reagent box that the invention discloses a kind of based on temperature change include manganese dioxide nano-plates, L-AA -2- tricresyl phosphate sodium salt, dopamine and buffer solution.The detection kit is irradiated reaction product using Portable near infrared laser, based on its temperature change, the thermometer or thermochromic patch of low cost can realize the Sensitive Detection to object, it can be applied to detection of alkaline phosphatase, there are the advantages such as easy, quick, selectivity is good, sensitivity is high.
Description
Technical field
The present invention relates to biosensor technique fields, more particularly to the alkaline phosphatase portable inspectiont based on temperature change
Kit and its application.
Background technique
In recent years, for detection object (such as albumen in spot or family and remote districts quickly, inexpensive
Matter, nucleic acid, small molecule and ion) portable sensor (no large-scale instrument and professional operator) research have become research heat
Point.The research and development of such product are of great importance for scientific research, clinical detection and bio-sensing field.With science
The fast development of technology, detecting (POCT) immediately, the fashionable whole world, many products have been commercialized.However, at present relatively
Mature portable sensor be it is very few, be also unable to satisfy the demand of people.Therefore, development is inexpensive, easy to operate
Portable detection sensor be very urgent.
Temperature testing equipment (thermometer, temperature paster etc.) is housed device common during we live, often based on these
The equipment seen is very significant by being simply designed to detection some diseases marker.Due to this personal value of these equipment
Lattice are cheap, easy to use, and do not need the talent of profession, are expected to be used for the POC detection of object.Also someone was developed in recent years
Some temperature sensors (thermometer) for being detected to disease marker and other objects, however there is also it is some not
Foot, such as need to use liposome embedding nir dye in advance, photothermal conversion ability is weak etc..Therefore, an operation letter is constructed
Single, without complicated modification, the temperature sensing platform of high sensitivity is very necessary.
Alkaline phosphatase is a kind of membrane bound enzyme, is present in biological tissue, and going for a variety of phosphate substrates can be catalyzed
Phosphorylation events, meanwhile, alkaline phosphatase is played the part of in the signal transduction and intracellular adjustment process in cell growth Apoptosis process
Drill key player.The unconventionality expression of blood plasma alkaline phosphatase is related with a variety of diseases, such as skeletal diseases, diabetes, and breast cancer,
Prostate cancer and dysfunction of liver etc..Therefore, develop sensitiveer, more preferably selecting property detection method and remove label alkaline phosphatase water
It is flat to be of great significance for the clinical diagnosis of a variety of diseases.And the method for traditional detection alkaline phosphatase activities includes isotope
Labelling method, it is complicated for operation, it is time-consuming inefficient, and need to endanger biggish radioisotopic tracer.Researcher creates one again
Pass through the different active new methods of analytical technology detection of alkaline phosphatase, such as chromatography, colorimetric method, chemoluminescence method, electricity a bit
Chemical method, spectroscopic methodology, serrs method.For colorimetric method although easy to operate, the sensitivity of detection is low,
Other methods come with some shortcomings although sensitivity is promoted, and such as need large-scale instrument, complicated surface modification and
Label etc..Therefore it is highly important for developing function admirable, portable analysis method for alkaline phosphatase activities detection.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a kind of alkalinity based on temperature change
Phosphatase portable detection reagent box, can be used the mode signal output easily and fast detected --- and temperature is detected, and is not necessarily to
Large-scale instrument and professional operator.Also provide that a kind of easy to operate, quick, portable, high sensitivity, selectivity is good and is not necessarily to
The method that professional training can quickly detect alkaline phosphatase.
In order to solve the above-mentioned technical problems, the present invention provides a kind of portable inspections of the alkaline phosphatase based on temperature change
Test agent box, including manganese dioxide nano-plates, L-AA -2- tricresyl phosphate sodium salt, dopamine and buffer solution.
Above-mentioned alkaline phosphatase portable detection reagent box, it is preferred that further include magnesium chloride.
Above-mentioned alkaline phosphatase portable detection reagent box, it is preferred that the buffer solution is Tris-HCl buffer.
Above-mentioned alkaline phosphatase portable detection reagent box, it is preferred that the concentration of the manganese dioxide nano-plates is 15 μ
The μ of g/mL~20 g/mL.
Above-mentioned alkaline phosphatase portable detection reagent box, it is preferred that the L-AA -2- tricresyl phosphate sodium salt
Concentration is 23 μ of μ g/mL~46 g/mL.
Above-mentioned alkaline phosphatase portable detection reagent box, it is preferred that the concentration of the dopamine is 20~40 μ g/
mL。
Above-mentioned alkaline phosphatase portable detection reagent box, it is preferred that the concentration of the magnesium chloride be 0.48mg/mL~
4.8mg/mL。
Above-mentioned alkaline phosphatase portable detection reagent box, it is preferred that the pH of the Tris-HCl buffer is 7.4.
Above-mentioned alkaline phosphatase portable detection reagent box, it is preferred that the alkaline phosphatase portable inspectiont examination
Agent box includes L-AA -2- tricresyl phosphate sodium salt, the 0.48mg/mL of the manganese dioxide nano-plates of 20 μ g/mL, 23 μ g/mL
The dopamine and Tris-HCl buffer of magnesium chloride and 20 μ g/mL.
As a total technical concept, the present invention also provides a kind of above-mentioned alkaline phosphatase portable detection reagents
Application of the box in detection of alkaline phosphatase.
Above-mentioned application, it is preferred that application method the following steps are included:
(1) manganese dioxide nano-plates and L-AA -2- tricresyl phosphate sodium salt are added in buffer and obtain detection examination
Agent；
(2) the alkaline phosphatase enzyme solutions of gradient concentration are added in the detection reagent and are incubated for；
(3) dopamine reaction reagent is added to be reacted；
(4) near infrared light is used, is changed with thermometer temperature collection, with the concentration of alkaline phosphatase enzyme solutions for horizontal seat
Mark draws standard curve by ordinate of the variation of temperature on thermometer；Alkaline phosphatase to be measured is calculated according to standard curve
Content；
Or, being managed near infrared light Ep, the variation of thermochromic patch on Ep pipe is observed, according to alkaline phosphatase standard
The concentration of sample draws standard color comparison card with the variation of thermochromic patch on Ep pipe, according to the standard color comparison card, with to
The resulting band of sample compares, and determines the alkaline phosphatase concentration of sample to be tested.
Above-mentioned application, it is preferred that described (2) are that the alkaline phosphatase enzyme solutions of magnesium chloride solution and gradient concentration are added
It is incubated in the detection reagent.
Above-mentioned application, it is preferred that the time being incubated in (2) are as follows: 30min~60min.Further, when incubation
Between be 40min.
Above-mentioned application, it is preferred that the time of reaction described in (3) is 20min.The reaction time is room temperature,
Specially 20~30 DEG C.
Above-mentioned application, it is preferred that the power of near infrared light described in (4) is 0.4W/cm2~1.0W/cm2；
The time of irradiation is 8min~20min, and the temperature when near infrared light is room temperature, specially 20~30 DEG C.Further
, the power of the near infrared light is 0.7W/cm2；The time of irradiation is 10min.
Compared with the prior art, the advantages of the present invention are as follows:
(1) the present invention provides a kind of alkaline phosphatase portable detection reagent box based on temperature change, by alkaline phosphorus
Sour enzyme, which is added in detection reagent, carries out catalysis reaction, if existing without alkaline phosphatase, manganese dioxide nano-plates can be catalyzed DOPA
Amine forms poly-dopamine, shows strong photothermal conversion ability；And contain alkaline phosphatase in sample to be tested, alkaline phosphatase energy
It is enough catalyzed L-AA -2- tricresyl phosphate sodium salt dephosphorylation, generates ascorbic acid.The ascorbic acid of generation can hydrolyze dioxy
Change manganese nanometer sheet, then can not form poly-dopamine nano particle, show as weak photothermal conversion ability.Utilize Portable near infrared
Laser is irradiated gained reaction product, is based on its temperature change, utilizes temperature sensor (such as thermometer or thermochromic
Patch) portable inspectiont to object can be realized, do not need the modification of other carrier embedding nir dye and complexity
Step belongs to a kind of novel signal acquisition behavior, does not need large-scale, complicated, expensive detecting instrument and equipment, utilizes letter
Single, low cost thermometer or thermochromic patch can realize the Sensitive Detection to object, and it is a kind of general for being expected to development
Portable temperature sensor.
(2) the application side of the present invention provides a kind of alkaline phosphatase portable detection reagent box based on temperature change
Method, this method do not need complicated modification step and professional's operation, do not need large-scale or expensive instrument and equipment, only yet
Only need a few step simplicity mixing and temperature detector (such as thermometer, thermochromic patch) if carry out the inspection of alkaline phosphatase
It surveys, detection mode letter becomes, is quick, portable, at low cost, while high specificity, and detection sensitivity is high.
Detailed description of the invention
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical scheme in the embodiment of the invention is clearly and completely described.
Fig. 1 is the detection method schematic illustration of alkaline phosphatase of the embodiment of the present invention.
Fig. 2 is in the embodiment of the present invention 1, and (temperature is counted as the testing result figure of the alkaline phosphatase enzyme standard solution of various concentration
For signal acquisition).
Fig. 3 is the testing result figure and standard colorimetric of the alkaline phosphatase enzyme standard solution of various concentration in the embodiment of the present invention 1
Block (thermochromic patch is as signal acquisition).
Fig. 4 is the influence result figure that different buffers detect alkaline phosphatase in the embodiment of the present invention 2.
Fig. 5 is the influence result that the dopamine solution of various concentration detects alkaline phosphatase in the embodiment of the present invention 3
Figure.
Fig. 6 is the shadow that the manganese dioxide nano-plates solution of various concentration detects alkaline phosphatase in the embodiment of the present invention 4
Ring result figure.
Fig. 7 is the influence result figure that the different enzymic catalytic reaction times detect alkaline phosphatase in the embodiment of the present invention 5.
Fig. 8 is the influence result figure that the magnesium ion of various concentration detects alkaline phosphatase in the embodiment of the present invention 6.
Fig. 9 is in the embodiment of the present invention 7, and the L-AA -2- tricresyl phosphate sodium salt (AAP) of various concentration is to alkaline phosphatase
The influence result figure of enzyme detection.
Figure 10 is the influence result that the different near infrared light times detect alkaline phosphatase in the embodiment of the present invention 8
Figure.
Figure 11 is the influence result that different near infrared light power detect alkaline phosphatase in the embodiment of the present invention 9
Figure.
Figure 12 is testing result figure of the varying environment temperature to alkaline phosphatase in the present invention in embodiment 10.
Figure 13 is specific detection result figure (thermometer and the thermochromic of different material in the present invention in embodiment 11
Patch).
Specific embodiment
Below in conjunction with Figure of description and specific preferred embodiment, the invention will be further described, but not therefore and
It limits the scope of the invention.
Material employed in following embodiment and instrument are commercially available.
Embodiment 1:
A kind of alkaline phosphatase portable detection reagent box of the invention based on temperature change, comprising: the two of 20 μ g/mL
Manganese oxide nanometer sheet, the L-AA -2- tricresyl phosphate sodium salt of 23 μ g/mL, 0.48mg/mL magnesium chloride and 20 μ g/mL DOPA
Amine.
A kind of application of the alkaline phosphatase portable detection reagent box of the present embodiment in detection of alkaline phosphatase, is answered
With method the following steps are included:
(1) manganese dioxide nano-plates and L-AA -2- tricresyl phosphate sodium salt are added in Tris-HCl buffer
((ingredient of Tris-HCl buffer are as follows: 10mmol/L Tris-HCl buffer, pH 7.4) obtains detection reagent, detection examination
Manganese dioxide nano-plates concentration is 20 μ g/mL in agent, and the concentration of L-AA -2- tricresyl phosphate sodium salt is 23 μ g/mL.
(2) prepare the standard solution of alkaline phosphatase: using Tris-HCl buffer as solvent, compound concentration is respectively
0.1U·L-1, 0.5UL-1, 1UL-1, 2UL-1, 5UL-1, 10UL-1, 20UL-1, 50UL-1, 100UL-1,
200U·L-1And 300UL-1Alkaline phosphatase enzyme solutions, room temperature preservation.
(3) the alkaline phosphatase enzyme solutions and magnesium chloride of various concentration are added in the detection reagent of step (1), are incubated for
40min, the concentration of magnesium chloride is 0.48mg/mL in detection reagent.
(4) dopamine is then added, concentration 20 μ g/mL of the dopamine in detection reagent is made, reacts 20min.
(5) near infrared light, (wavelength of near infrared light is 780nm, and power is 0.7W/cm2, laser is common near-infrared
Laser or Portable near infrared laser) irradiation 10min, change or observe thermochromic on Ep pipe with thermometer temperature collection
The variation of patch (i.e. temperature discoloring paster).If its testing principle exists without alkaline phosphatase as shown in Figure 1:, manganese dioxide is received
Rice sector-meeting catalysis dopamine forms poly-dopamine, shows strong photothermal conversion ability；And contain alkaline phosphatase in sample to be tested
Enzyme, alkaline phosphatase can be catalyzed L-AA -2- tricresyl phosphate sodium salt dephosphorylation, generate ascorbic acid.What is generated is anti-bad
Hematic acid can hydrolyze manganese dioxide nano-plates, then can not form poly-dopamine nano particle, show as weak photothermal conversion ability.
Using the concentration of alkaline phosphatase enzyme solutions as abscissa, it is bent that standard is drawn using the variation of temperature on thermometer as ordinate
Line, the regression equation of standard curve: y=0.774x+0.039, coefficient R2=0.9969.
Fig. 2 is the testing result figure of the alkaline phosphatase enzyme standard solution of various concentration (thermometer is as signal acquisition)
Alternatively, drawing standard according to the concentration of alkaline phosphatase standard sample with the variation of thermochromic patch on Ep pipe
Colorimetric card.
Fig. 3 is the testing result figure and standard color comparison card (thermochromic patch of the alkaline phosphatase enzyme standard solution of various concentration
As signal acquisition).
(6) solution to be measured and magnesium chloride are added in the detection reagent of step (1), are incubated for 40min, then add 20
μ g/mL dopamine reaction reagent, reacts 20min at room temperature.With near infrared light, (wavelength of near infrared light is 780nm, and power is
0.7W/cm2, laser is common near infrared laser or Portable near infrared laser) and irradiation 10min, according to standard curve
Calculate the content of alkaline phosphatase.
Alternatively, to compare with the resulting band of sample to be tested, determining the alkali of sample to be tested according to this standard color comparison card
Acid phosphatase concentration.
Embodiment 2:
Investigate influence of the different buffer solutions to detection effect:
Manganese dioxide nano-plates and L-AA -2- tertiary sodium phosphate salt are added separately to phosphate buffer, and (phosphoric acid is slow
The ingredient of fliud flushing are as follows: 10mmol/L phosphate buffer, pH 7.4, the uniform component of phosphate buffer causes in full text),
Tris-HCl buffer (ingredient of Tris-HCl buffer are as follows: 10mmol/L Tris-HCl buffer, pH 7.4, in full text
The uniform component of phosphate buffer causes), HEPES buffer solution (10mmol/L HEPES buffer, pH 7.4, HEPES in full text
The uniform component of buffer causes) in, it is detected according to the method for embodiment 1.
Testing result is as shown in Figure 4: temperature change value is maximum in Tris-HCl buffer, and signal is most strong.Therefore, subsequent
Tris-HCl buffer is chosen in embodiment as optimized buffer liquid.
Embodiment 3:
Investigate influence of the dopamine solution concentration to detection effect:
Dopamine is dissolved in Tris-HCl buffer, the dopamine solution of various concentration: 0 μ g/mL, 5 μ g/ is prepared
ML, 10 μ g/mL, 15 μ g/mL, 20 μ g/mL, 30 μ g/mL and 40 μ g/mL.The step of according to embodiment 1, various concentration is added
Dopamine solution is detected.
Testing result is as shown in Figure 5: reaching platform, temperature when dopamine concentration is 20~40 μ g/mL in detection reagent
Changing value is maximum, and signal is most strong.Therefore, it is best dopamine solution concentration that 20 μ g/mL are chosen in subsequent embodiment.
Embodiment 4:
Investigate influence of the manganese dioxide nano-plates concentration to detection effect:
Manganese dioxide nano-plates and L-AA -2- tricresyl phosphate sodium salt are added in Tris-HCl buffer and are configured to
The detection reagent of various concentration, wherein the concentration of manganese dioxide nano-plates is respectively 0 μ g/mL, 5 μ g/mL, 10 μ g/mL, 15 μ g/
ML and 20 μ g/mL, the concentration of L-AA -2- tricresyl phosphate sodium salt are 23 μ g/mL.It is examined according to the method for embodiment 1
It surveys.
Testing result is as shown in Figure 6: when manganese dioxide nano-plates concentration is 20 μ g/mL, temperature change value is maximum, signal
It is most strong.So choosing 20 μ g/mL is the manganese dioxide nano-plates concentration in subsequent embodiment.
Embodiment 5:
Investigate influence of the enzymic catalytic reaction time to detection effect:
Contain manganese dioxide nano-plates and L-AA -2- phosphoric acid according to solution to be measured is added the step of embodiment 1
In the Tris-HCl buffer of trisodium salt, respectively react 0min, 5min, 10min, 20min, 30min, 40min, 50min and
60min investigates influence of the temperature change to testing result.
Testing result is as shown in Figure 7: with the extension of reaction time, temperature change value gradually rises, when reaching 40min,
Temperature change value reaches platform, and signal is most strong.So choosing 40min is the best enzymic catalytic reaction time in subsequent embodiment.
Embodiment 6:
Investigate influence of the magnesium ion concentration to detection effect:
Solution to be measured is added in detection reagent in step (6), while being added the magnesium ion of various concentration: 0.00mg/mL,
0.12mg/mL, 0.24mg/mL, 0.48mg/mL, 0.96mg/mL, 1.92mg/mL and 4.8mg/mL react 40min.Remaining step
It is rapid same as Example 1.
Testing result is as shown in Figure 8: with the increase of magnesium ion concentration, temperature change value is also promoted therewith, and when magnesium from
When sub- concentration is higher than 0.48mg/mL, reach platform.Therefore, it is best in subsequent embodiment for choosing 0.48mg/mL (5mmol/L)
Magnesium ion concentration.
Embodiment 7:
Investigate influence of the L-AA -2- tertiary sodium phosphate salinity to detection effect:
Manganese dioxide nano-plates and L-AA -2- tricresyl phosphate sodium salt are added in Tris-HCl buffer and are configured to
The detection reagent of various concentration, wherein the concentration of manganese dioxide nano-plates is 20 μ g/mL, L-AA -2- tertiary sodium phosphate
The concentration of salt is respectively 2.87 μ g/mL, 5.75 μ g/mL, 11.5 μ g/mL, 23 μ g/mL, 34.5 μ g/mL and 46 μ g/mL.It will be to be measured
The magnesium ion that solution and concentration are 5mmol/L is added in detection reagent, is detected according to the method for embodiment 1.
It is as shown in Figure 9 to investigate result: with the increase of L-AA -2- tertiary sodium phosphate salinity, temperature value variation
Increase therewith, when L-AA -2- tertiary sodium phosphate salinity is greater than 23 μ g/mL, temperature change value reaches platform.So choosing
Taking 23 μ g/mL is the L-AA -2- tertiary sodium phosphate salinity in subsequent embodiment.
Embodiment 8:
Investigate influence of the near-infrared light application time to detection effect:
In step (6), under near infrared light, (wavelength of near infrared light is 780nm, and power is 0.7W/cm2, laser
Device is common near infrared laser or Portable near infrared laser), irradiate respectively 0min, 2min, 4min, 6min, 8min,
10min, 15min and 20min；Changed with monitors temperature.Remaining step is same as Example 1.
The results are shown in Figure 10 for investigation: as the irradiation time increases, temperature change value gradually rises, when reaching 10min,
Temperature change value reaches platform, and signal is most strong.So when selection 10min is the best near infrared light in subsequent embodiment
Between.
Embodiment 9:
Investigate influence of the near infrared light according to power to detection effect:
In step (6), with the near infrared light (0.4W/cm of different capacity2、0.5W/cm2、0.6W/cm2、0.7W/cm2、
0.8W/cm2、0.9W/cm2、1.0W/cm2) irradiation 10min；Changed with monitors temperature.Remaining step and implementation
Example 1 is identical.
It is as shown in figure 11 to investigate result: when power is 0.7W/cm2When, temperature change value is maximum, and signal is most strong.Therefore, it selects
Take 0.7W/cm2As near infrared light power optimal in subsequent embodiment.
Embodiment 10:
Investigate influence of the varying environment temperature to detection effect:
In step (6), 10min is irradiated under near infrared light in different temperature (20~30 DEG C)；With with temperature
The variation of sensor monitoring temperature.Remaining step is same as Example 1.
It is as shown in figure 12 to investigate result: temperature influences very little to alkaline phosphatase detection effect at 20~30 DEG C.Therefore, should
Invention can be used at 20~30 DEG C.
Embodiment 11: specificity is investigated
A kind of alkaline phosphatase portable detection reagent box of embodiment 1 specific investigation side in detection of alkaline phosphatase
Method, the present embodiment are same as Example 1 using the detection reagent and near-infrared exciter of alkaline phosphatase.
Specific specificity investigation method the following steps are included:
(1) configure different material (including enzyme and albumen) standard solution, as in the present embodiment specificity investigate to
Surveying solution has: horseradish peroxidase (HRP), glucose oxidase (Gox), glucoamylase (GA), lysozyme (LZM), C
Reactive protein (CRP) and bovine serum albumin(BSA) (BSA), wherein enzyme concentration is 20U/L, and the concentration of albumen is 2mg/mL.
(2) above-mentioned solution to be measured is added separately to detect in the reaction system of condition same as Example 1, is recorded
Temperature change at this time and the color change for observing Ep pipe thermochromic patch, as a result as shown in figure 13.
It is from the testing result of Figure 13 it can be seen that horseradish peroxidase, glucose oxidase, glucoamylase, molten
Bacterium enzyme, c reactive protein, bovine serum albumin(BSA) have little effect inspection result, only after alkaline phosphatase is added, temperature
Difference can just show, and Ep pipe thermochromic patch color can just change, and thus prove the method for the present embodiment to alkaline phosphatase
There is good specificity.
The above described is only a preferred embodiment of the present invention, being not intended to limit the present invention in any form.Though
So the present invention is disclosed as above with preferred embodiment, and however, it is not intended to limit the invention.It is any to be familiar with those skilled in the art
Member, in the case where not departing from Spirit Essence of the invention and technical solution, all using in the methods and techniques of the disclosure above
Appearance makes many possible changes and modifications or equivalent example modified to equivalent change to technical solution of the present invention.Therefore,
Anything that does not depart from the technical scheme of the invention are made to the above embodiment any simple according to the technical essence of the invention
Modification, equivalent replacement, equivalence changes and modification, all of which are still within the scope of protection of the technical scheme of the invention.
Claims (10)
1. a kind of alkaline phosphatase portable detection reagent box based on temperature change, which is characterized in that received including manganese dioxide
Rice piece, L-AA -2- tricresyl phosphate sodium salt, dopamine and buffer solution.
2. alkaline phosphatase portable detection reagent box according to claim 1, which is characterized in that further include magnesium chloride.
3. alkaline phosphatase portable detection reagent box according to claim 2, which is characterized in that the buffer solution is
Tris-HCl buffer.
4. alkaline phosphatase portable detection reagent box according to claim 3, which is characterized in that the manganese dioxide is received
The concentration of rice piece is 15 μ of μ g/mL~20 g/mL；
And/or the concentration of the L-AA -2- tricresyl phosphate sodium salt is 23 μ of μ g/mL~46 g/mL；
And/or the concentration of the dopamine is 20 μ of μ g/mL~40 g/mL；
And/or the concentration of the magnesium chloride is 0.48mg/mL~4.8mg/mL；
And/or the pH of the Tris-HCl buffer is 7.4.
5. alkaline phosphatase portable detection reagent box according to claim 1, which is characterized in that the alkaline phosphatase
Enzyme portable detection reagent box includes the L-AA -2- tertiary sodium phosphate of the manganese dioxide nano-plates of 20 μ g/mL, 23 μ g/mL
The dopamine and Tris-HCl buffer of salt, the magnesium chloride of 0.48mg/mL and 20 μ g/mL.
6. alkaline phosphatase portable detection reagent box described in a kind of any one of claims 1 to 5 is in detection alkaline phosphatase
Application in enzyme.
7. application according to claim 6, which is characterized in that application method the following steps are included:
(1) manganese dioxide nano-plates and L-AA -2- tricresyl phosphate sodium salt are added in buffer and obtain detection reagent；
(2) the alkaline phosphatase enzyme solutions of gradient concentration are added in the detection reagent and are incubated for；
(3) dopamine reaction reagent is added to be reacted；
(4) near infrared light is used, is changed with thermometer temperature collection, using the concentration of alkaline phosphatase enzyme solutions as abscissa, with
The variation of temperature is that ordinate draws standard curve on thermometer, calculates containing for alkaline phosphatase to be measured according to standard curve
Amount；
Or, being managed near infrared light Ep, the variation of thermochromic patch on Ep pipe is observed, according to alkaline phosphatase standard sample
Concentration, standard color comparison card is drawn with the variation of thermochromic patch on Ep pipe, according to the standard color comparison card, and to test sample
The resulting band of product compares, and determines the alkaline phosphatase concentration of sample to be tested.
8. application according to claim 7, which is characterized in that described (2) are by the alkali of magnesium chloride solution and gradient concentration
Acid phosphatase solution is added in the detection reagent and is incubated for.
9. application according to claim 7 or 8, which is characterized in that the time being incubated in (2) are as follows: 30min~
60min；
And/or the time of reaction described in (3) is 20min.
10. application according to claim 7 or 8, which is characterized in that the power of near infrared light described in (4)
For 0.4W/cm2~1.0W/cm2；The time of irradiation is 8min~20min；Environment temperature is 20~30 DEG C.
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| CN112816414A (en) * | 2021-01-07 | 2021-05-18 | 武汉大学 | Alkaline phosphatase detection kit based on dual-emission lanthanide MOF and detection method |
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